Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC152
Caveolae Limit β2 Adrenoceptor-derived cAMP Signals in the Adult Cardiac Myocyte
D. A. MacDougall1, J. A. Collins1, H. Chu1, J. Colyer1, S. C. Calaghan1
1. Institute of Membrane and Systems Biology, University of Leeds, Leeds, United Kingdom.
Stimulation of β1-adrenoceptors (ARs) produces marked positive inotropic and lusitropic responses in the cardiac myocyte, which are absent following β2-AR stimulation. Compartmentation of β2-AR-derived cAMP-PKA signals is thought to contribute to this functional discrepancy. We are interested in how caveolae (sarcolemmal invaginations rich in cholesterol, sphingolipids and caveolin-3) contribute to this compartmentation in the adult rat ventricular myocyte (ARVM). We have previously observed substantial augmentation of β2-AR-mediated inotropic and lusitropic responses following cholesterol depletion of ARVM (1). Here, we (i) confirm the specificity of this effect on caveolae by multiple approaches and (ii) investigate which proteins could play a role via residence in caveolae. ARVM were treated with either 1 mM methyl-β-cyclodextrin (MBCD) to deplete cholesterol, or conjugated MBCD:cholesterol (molar ratio 1:8). In separate experiments, some cells were incubated with 0.5 μM TAT-C3SD (a cell-permeable peptide inhibitor of caveolin-3 scaffolding interactions) or TAT-Scram (scrambled sequence). β2-AR stimulation was achieved with 10 µM zinterol in the presence of 300 nM CGP20712A; cell shortening was measured with edge-detection software. Protein and phospho-protein levels were determined by immunoblotting. ARVM were fractionated on a discontinuous sucrose gradient. Statistical significance was assessed with the Student’s t-test. Following β2-AR stimulation, both shortening and time to half relaxation were enhanced in MBCD-treated compared to control cells (70.2 ± 9.7% vs 4.9 ± 5.8% and -13.3 ± 1.3% vs -5.4 ± 1.3% respectively; P<0.001, n=13-20). These inotropic and lusitropic effects of MBCD were greatly attenuated by 87 and 77% respectively when MBCD was conjugated to cholesterol prior to treatment of cells (P<0.01, n=11-13). We have previously linked MBCD effects to a selective increase in protein kinase A (PKA) phosphorylation of phospholamban at Ser16 (pPLB), but not troponin I at Ser23/24 (pTnI) (1). Pre-incubation of ARVM with TAT-C3SD peptide also enhanced pPLB during β2-AR stimulation compared to that with TAT-Scram (46.1 ± 6.3-fold vs 2.2 ± 0.8-fold respectively; P<0.01, n=3). Levels of pTnI were unaltered by the same treatments (P>0.05, n=3). Finally, we detected caveolin-3, β2-AR, Gαi3, adenylyl cyclase 5/6, PKA RII, protein phosphatase 2a, and G-protein-coupled receptor kinase, but not phosphodiesterase 3A or 4D, in caveolar raft fractions. We conclude that in ARVM (i) the effects of MBCD on β2-AR stimulation are specific to cholesterol depletion and caveolae disruption and (ii) the signal components resident in caveolae have the potential to compartmentalise cAMP by facilitating processes that limit the magnitude and propagation of cAMP signals, including β2-AR phosphorylation and β2-AR -Gi coupling.
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